IPST Technical Paper Series Number 605

Comparison of the Properties of Native and Pentaammineruthenium(!ll)-modified Xylanase

B.R. Evans, L.M. Lane, R. Margalit, G.M. Hathaway, A. Ragauskas, and J. Woodward

January 1996

Submitted toEnzyme and Microbiology Technology

Copyright® 1996by the Instituteof PaperScienceand Technology

For Members Only

COMPARISON OF THE PROPERTIES

OF NATIVE AND PENTAAMMINERUTHENIUM(III)-MODIFIED XYLANASE

Barbara R. Evans, _'¢ Lynette M. Lane, a Ruth Margalit, b Gary M. Hathaway, _Arthur Ragauskas, a and Jonathan Woodward _

a Oak Ridge National Laboratory _Oak Ridge, TN 37831-6194

b Jet Propulsion Laboratory 2Pasadena, CA 91109

° California Institute of TechnologyBeckman Institute

Pasadena, CA 91125

dlnstitute of Paper Science and TechnologyAtlanta, GA 30318

Corresponding author

'q'he submitted manuscript has been authored by acontractor of the U. S. Government under contract No.

DE-AC05-84OR21400. Accordingly, the U. S.

Government retains a nonexclusive, royalty-freelicense to publish or reproduce the published: form ofthis contribution, or allow others to do so, for U. S.Government purposes."

'Managed by Lockheed Martin Energy Systems, Inc., for the U.S. Department of Energyunder contract DE-AC05-84OR21400.

2Operating Division of California Institute of Technology under contract NAS7-918 withthe National Aeronautics and Space Administration.

Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were

purified, then modified by the attachment of pentaammineruthenium, resulting in the

generation of a xylanase with veratryl alcohol oxidase activity. Hydrolytic activity of

T. reesei xyn II on soluble xylans was unchanged by modification with

pentaammineruthenium; however, modification of B. pumilus xynA greatly reduced

xylan hydrolysis unless the active site of the xylanaSe was protected with xylose during

the modification. The presence of histidine, cysteine, or reduced glutathione during

xylan hydrolysis greatly increased the xylanase activity of the pentaammineruthenium-

modified B. pumilus xylanase. Glycine, glutamic acid, methionine, or oxidized

glutathione had no effect on xylanase activity.

keywords:

chemical modification/xytanase/mthenium/oxidase

Introduction

Ruthenium complexes generate hydrogen peroxide, superoxide, and hydroxyl

radicals by reaction with molecular oxygen. _'2Pentaammineruthenium can be attached to

proteins by coordination of the metal to a surface-accessible histidine residue of the protein

as a sixth ligand. Attachment of pentaamineruthenium(III) imparts unique redox properties

to myoglobin, azurin, and cytochrome c without perturbing their structure significantly. 3-5

Attachment of pentaammineruthenium to cellobiohydrolase t and II (CBH I and II), the

main cellulases of the fungus Trichoderma reesei, enables the enzymes to oxidize veratryl

alcohol, a substrate of lignin peroxidase, an oxidoreductase activity not present in the

native cellobiohydrolases. 64

Xylanases are used to decrease the mount of chemicals required in the bleaching

of wood pulp for paper manufacture. Improved efficacy of xylanase pretreatment would

further decrease the use of bleaching chemicals and improve the results from "totally

chlorine-free" bleaching. Hardwood xylans, such as birchwood xylan, are composed of

acetyl-4-O-methylglucuronoxylan with a degree of polymerization (DP) of about 200, and

with one in ten of the [_-D-xylopyranoside backbone units having a 1,2-1inked 4-0-

methylglucuronic acid moiety. The xylan found in cereals, such as oat spelt xylan, and

other grasses is arabino-4-methyl-glucuronoxylan with a DP of about 70 and with L-

arabinofuranosyl side chains. 9 The differences between the two types of xylan can affect

the bleaching of different wood pulps. _° The xylanase pretreatment aids the removal of

lignin from the wood pulp by hydrolyzing the xylan backbone of the hemicellulose to

which the lignin is covalently attached. If the xylanase were equipped with a redox group,

the bifunctional enzyme thus designed might be able to directly attack and depolymerize

the lignin in a manner similar to that employed by lignin peroxidases. In order to design

a bifunctional xylanase, we modified two xylanases, xynA from Bacillus pumilus and xyn

II from Trichoderma reesei, by attachment of pentaammineruthenium groups. Both of these

xylanases are small proteins with alkaline pi (molecular weight 22,000 and 21,000, and pis

9.3 and 9.0, respectively). They belong to the G family of xylanases, a group of

homologous small xylanases of alkaline pi that have been isolated from various fungi and

bacteria. 9'_1 The bacterial xylanase, xynA, possesses four histidine residues, at positions 11,

59, 60, and 160 from the amino terminus of the protein; it also has one cysteine residue at

position 82. The fungal xylanase xyn II has histidines at positions 22, 144, and 155 from

the amino terminus. The catalytically active residues of these xylanases had been reported

to be two glutamic acids, at positions 93 and 182 for xynA, 12 and at 86 and 177 for xyn

11.13 Histidine had not been reported to be a catalytically active residue in xylanases, 14q7

so no loss of hydrolytic activity due to attachment of pentaammineruthenium was

anticipated. The properties of the modified xylanases are reported here.

Materials and methods

Purification and modification of enzymes

Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were

purified from commercially available crude culture filtrates (Pulpzyme HB TM and SP431,

formerly Pulpzyme HA TM, generous gifts from NovoNordisk Biotech, Inc., Danbury, CT).

Purification stages of the proteins were analyzed by sodium dodecyl sulfate (SDS)

and isoelectric focusing polyacrylamide gels.

To purify the B. pumilus xylanase, 10-mi aliquots of culture filtrate were applied

to 5 x 50 cm columns of Biogel P-30 (BioRad) in 5 mM ammonium carbonate, pH 7.8.

Protein fractions from the P-30 chromatography were applied to 1.0 x 9 cm columns of

DEAE Sephadex in the same buffer. The purified xylanase appeared homogeneous on

SDS gels (Figure 1). Xylanase samples were lyophilized, and the dry protein was used for

the modification with pentaammineruthenium(II).

The Trichoderma culture filtrate was found to contain ali of the Trichoderma

cellulases, in addition to the two xylanases. The crude filtrate (30 ml) that had been

dialyzed overnight at 4°C agmst 5 mM ammonium carbonate, pH 7.8, was applied to a 50-

ml column of DEAE Sephadex in 5 mM ammonium carbonate, pH 7.8. At this pH,

xylanase II, with a pi of 9.0, should not bind to the ion-exchange matrix, whereas the

cellulase components should bind to the DEAE Sephadex. About 5-10% cellulase

(cellobiohydrolase II, endoglucanases I and II) was present in the eluted xylanase II

fraction....

Attachment of pentaammineruthenium to the xylanases was carried out as described

previously for CBH I. 6

Analytical methods

The presence of pentaammineruthenium attached to histidine was determined

spectrophotometrically by the appearance of an absorption peak at 360 nm and by the

appearance of a peak at 110 mV in square-wave voltammograms. _8 The ratio of Ru(NH3)s-

histidine to xylanase was calculated from the absorbance of the modified xylanase at 360

nmand the molar absorption coefficient of Ru(NH3)5-histidine (3000 M -_ cm-_), and from

the absorbance of the protein at 280 nm, using a molar absorption coefficient (56,000 M -_

cm -_) that was calculated from the tryptophan and tyrosine content of the protein. _2

Polyacrylamide gel electrophoresis was carried out with a Pharmacia Biotech

Phasff M system using Pharmacia Biotech precast IEF 3-9 and 10-15 gradient gels.

Pharmacia Biotech low-molecular-weight markers and IEF 3-10 markers were prepared and

used according to the manufacturer's instructions. Samples for SDS electrophoresis were

incubated with 2% SDS and 5% [3-mercaptoethanol for 5 min at 90°C before loading them

on the gels.

Assays of enzymatic activity

Oxidoreductase activity was assayed with the lignin peroxidase substrate veratryl

alcohol. 7'_9 Xylanase assays were carried out at 45°C in 50 mM sodium acetate, pH 6.0

(pH 5.0 for the Trichoderma xylanase) with 1% birchwood or oat spelt xylan and 0.010

mg/ml xylanase. Xylanase activity was assayed by measuring reducing sugar production

with the dinitrosalicylic acid reagent. 2° In determining the optimal pH for the B. pumilus

enzyme, 50 mM sodium phosphate buffer was used for the assays at pH 7.0, and 50 mM

Tris-HC1 buffer was used for the assays at pH 8.0, 8.5, or 9.0.

High-pressure liquid chromatography (HPLC) analysis

Purification of xylanase samples prior to tryptic digestion as well as fractionation

of tryptic peptides was carried out with a Perkin-Elmer Applied Biosystems Model 172

microbore liquid chromatography system. For reverse-phase chromatography, a 1.0 x 100

mm Nucleosil C18 column and a 0.5 x 150 mm Reliasil C18 column (Column Engineering,

Ontario, CA) were run using a gradient of 2% acetonitrile/0.05% trifluoroacetic acid to

90% acetonitrile/0.045% trifluoracetic acid, with a flow rate of 0.025 or 0.06 ml/min,

depending on column diameter. Whole xylanase was detected by monitoring at 280 nm;

tryptic peptides, by monitoring at 216 and 305 nm.

Tryptic digests

Samples (1.0 mg/ml) of native and modified xylanase were purified by C 18 reverse-.

phase HPLC before digestion with trypsin. Purified xylanase was dried and then

redissolved in 0.020 ml of 0.2 M ammonium bicarbonate, pH 8.5, to a final reaction

L

volume of 0.030 mi. Then 2 gl of a solution of 1 mg/ml modified trypsin (Promega) was

added, and digestion was allowed to proceed for 12 h at ambient temperature. Digestion

was terminated by the additionof 0.005 m! trifluoroacetic acid, followed by drying the

sample in vacuo.

Mass spectrometry and peptide sequencing

Analysis by mass spectrometry and peptide sequencing was carded out at the Protein

and Peptide Micro Analytical Facility, which is located ai the Beckman Institute, the

California Institute of Technology, Pasadena, California.

Matrix-assisted, time-of-flight (MALDI TOF) mass spectrometry was carried out

with a Lasertech II mass spectrometer (Perseptive Biosystems, Cambridge, MA), equipped

with a reflector, a 1.3-m flight tube, and a nitrogen laser. HPLC fractions (0.5 _1) were

spotted to the sample slides with sinapinic acid (10 mg/ml) and ec-cyano-4-hydroxycinnamic

acid. Acetylangiotensinogen (MW 1801.09) and bovine cytochrome c (MW 12,360.1)

were used as internal and external calibrams.

Peptides were identified by Edman degradation with a model 476 automated

microsequencer (Perkin Elmer/Applied Biosystems, Foster City, CA) and comparison with

the known sequence. The coupling buffer was N-methyl-piperidine, and the samples were

spotted to Porton peptide disks (Porton Instruments Division, Beckman Instruments,

Oxnard, CA).

Results

Purification of xylanases

The bacterial xylanase xynA was reportedly produced in a bacterial strain without

endogenous cellulases and was apparently homogenous, simplifying its purification. The

majority of the modification and pulping experiments were therefore carried out with this

xylanase. Contaminants from the culture media were removed by gel filtration on Biogel

P-30 in 5 mM ammonium carbonate, pH 7.8, followed by ion-exchange chromatography

on DEAE Sephadex in the same buffer (Figure 1).

In the case of the T. reesei xylanase xyn II, _3the culture filtrate obtained from

NovoNordisk contained the entire multicomponent cellulase system of the fimgus with xyn

II making up only about 10% of total protein. The relative amount of xylanase is

increased, but the cellulases are still present in large amounts, 21 necessitating purification

with multiple ion-exchange chromatography steps. To evaluate the suitability of this

enzyme, modification of this partially purified xylanase preparation was carried out. The

attachment of pemaarraninemthenium to xyn II did not adversely affect its xylanase activity

to the same degree as observed in the case of the bacterial xylanase described above. The

hydrolytic activity of pentaammineruthenium-modified xyn II towards birchwood and oat

l0

spelt xylans remained fairly close to that of the native control (modified, 162 and 168 _tmol

mg -_ min -_; native, 207 and 190 _tmol mg -_ min-1).

Properties of pentaammineruthenium(III)-modified xynA

The effect of modification with pentaammineruthenium upon the xylanase xynA

from Bacillus pumilus depended upon the modification conditions. Modification was

carried out for 2 or 4 h with a tenfold excess of aquopentaammineruthenium(II). The

properties of the xylanase after attachment of pentaamineruthenium were the same for the

2- and 4-h modification times, but attachment was found to be more reliable when a 4-h

modification time was used. A substantial loss of activity in hydrolysis of birchwood and

oat spelt xylans was observed for xynA modified with pentaammineruthenium. Loss of

activity was reduced when xynA was modified in the presence of 0.10 M xylose to protect

the active site of the enzyme (Table 1). Veratryl alcohol oxidase activity was higher for

xynA modified without xylose, and the number of pentaammineruthenium groups attached

to the xylanase, based on the absorption peak at 360 nm from the pentaammineruthenium-

histid'me, was 2 to 3, as opposed to 1 for enzyme protected with xylose.

The optimal pH for hydrolysis of birchwood xylan was not changed by the

attachment of pentaamminemthenium. For both native and modified xynA, maximum:.

hydrolysis was observed at pH 6.0 (Figure 2).

11

Effect of _ee amino acids on xylanase activity

When the inactivated xylanase was incubated with 50 mM histidine at pH 6.0,

activity towards xylan was partially restored. No restoration of activity occurred when the

modified xylanase was incubated with 50 mM glycine, implying that the imidazole group

of the histidine was required for restoration of activity (Figure 3). When the xylanase was

incubated with 200 mM histidme for 18 h at 4°C and then filtered on Sephadex G-25 to

remove excess histidine, the absorption peak at 360 nm from Ru(NH3)5-histidine

disappeared (data not shown). Since the absorption peak at 360 nm is characteristic of

pentaamminemthenium-histidine, its disappearance implies that the ruthenium complex was

displaced from the histidine on the xylanase by the excess free histidine. Further

investigations of the effects of various amino acids upon the xylanase activity of

inactivated, pentaammineruthenium-modified xynA are summarized in Table 2. Amino

acids were added directly to control and enzyme reaction mixtures containing 1%

birchwood xylan, and the net production of reducing sugar was determined for the reaction

containing enzyme. At the concentrations used in this study (5-50 mM), histidine doubled

the specific activity of the inactivated xylanase, but reduced glutathione and cysteine

increased it fourfold to fivefold. Oxidized glutathione, glutamic acid, glycine, and

methionine had no effect on the activity of the xylanase (Figure 3 and Table 2). Control

reactions in which xylan was omitted showed no product from the action of the modified

xylanase on cysteine, or on reduced or oxidized glutathione, which gave a reaction with the

12

dinitrosalicylic acid reducing sugar reagent.

Determination of site of attachment of pentaammineruthenium(lll)

To determine the site of attachment of the pentaammineruthenium on the mottled

xynA, Edman sequence determination and mass spectroscopy were carried out in tandem

on whole native and modified xynA and on tryptic peptides prepared from the two proteins.

Before further analysis by mass spectroscopy and tryptic digestion was carried out,

native and pentammineruthenium(I!I)-modified xynA were purified by reverse-phase HPLC

on Nucleosil C18. Mass spectroscopy of the HPLC-purified proteins gave a mass value for

native, unmodified xylanase of 22,577 + 25 amu and a mass value for

pentaammineruthenium(III)-modified xynA of 22,950 + 50 amu. The increase in mass of

370 amu observed for modified xylanase corresponds to that expected for the addition of

two pentaamineruthenium groups (mass 186) to the protein.

Native and pentaamminemthenium(III)-modified xynA were sequenced directly from

the amino terminus using Edman degradation. Peptide sequencing of the whole protein of

pentaammineruthenium(III)-modified xynA identified the first 20 amino acid residues and

determined that the ruthenium complex had not been attached to H11. The N-termin_

amino acid sequence was observed to differ from the published sequence of xynA from

Bacillus pumilus:

published sequence: RTITNNEMGNHSGYDYELWKD

13

observed sequence: ETIYDNRIGTHSGYDFELWKD

Another six differences between the published sequence and the observed sequence were

noted: N44 was changed to K, A159 was changed to E, R161 was missing, M173 was

changed to S, Q184 was changed to S, and S186 was changed to K. Overall, the percent

identity between the published sequence _7 and the sequence determined from the

NovoNordisk Pulpzyme HB TM xylanase was 93%. It appears that the NovoNordisk enzyme

may be isolated from a different strain of Bacillus pumilus than that used in the published

sequence determination.

The tryptic peptides generated from native and modified xylanase were compared

by analysis with HPLC and mass spectroscopy, a method that has been used to determine

the site of attachment of substrate analogs to exoglucanases and [3-glucosidases. 22 Tryptic

digests were carried out on native and modified xynA as described above. The tryptic

peptides were £ractionated by reverse-phase HPLC. The HPLC effluent was monitored at

216 nm to detect peptides and at 305 nm to detect the presenc e of pentaammineruthenium-

histidine (absorbance maximum is shifted to 305 nm at acid pH). When detection was

carried out at 216 nm, a peak present in the column profile of the tryptic digest of native

xylanase at 32.5 min was absent from the chromatogram of the pentaammineruthenium(III)-

modified xylanase (Figure 5). This peak, missing from the tryptic digest of the modified

xylanase, was found to contain several peptides, including one with a mass of 1279.5 amu.

The peptides present in the peak were sequenced, and the peptide of mass 1279.5 was

determined to correspond to residues 151-162 of the published sequence.

14

To establish that the peptide corresponding to 151-162 of the published sequence

was the site of attachment of pentaammineruthenium, the chromatogram of the tryptic

peptides from the modified xylanase was examined for the presence of a peptide

corresponding to the mass of the missing peptide plus one or two pentaammineruthenium

groups. When the chromatogram of the tryptic digest of modified xylanase was monitored

at 305 nm, several peaks were detected. One peak, which eluted at 31 min in the HPLC

chromatogram, had a high ratio of absorbance at 305 nm to absorbance at 216 nm,

indicating that it was likely to contain pentaammineruthenium-modified peptides. The

presence of a peptide with mass of 1650.8 was detected in the peak at 31 min from the

tryptic digest of the modified xylanase. The mass of this peptide (1279.5 amu)

corresponds to the mass of the native peptide consisting of residues 151-162, plus a mass

equivalent to two pentaammineruthenium groups. Sequence determination by Edman

degradation confirmed the identity of the peptide present in the peak eluting at 31 min as

corresponding to residues 151-162 of the xylanase'

TSGTVSVSEHFK

No tryptic peptides of mass 1650.8 were detected in the digest of the native xylanase. This

implies that the pentaammineruthenium is attached to the 151-162 peptide, which contains

one histidine at position 160. The location of the attached pentaammineruthenium thus

appears, by a process of elimination, to be the histidine at position 160, an amino acid

residue that is found to be conserved among the family G xylanases. _3 The second

pentaammineruthenium group could be attached to the glutamic acid residue adjacent to the

%

15

histidine on the same peptide.

Determination of kinetic parameters for native and pentaammineruthenium-modified xynA

To investigate the effect of attachment of pentaammuinemthenium to xynA, the

kinetic parameters of native and 4-h pentaammineruthenium-modified xynA were

determined for the hydrolysis of birchwood xylan (Figure 6 A and B). The Km values for

native and modified xynA were determined to be 6.44 and 3.76 mg/ml birchwood xylan,

respectively. For native and modified xynA, the Vma x values were 464 and 141 mM

reducing sugar min -_ (mg xylanase) -_, respectively. Loss of activity upon modification

would thus appear to result from the inability of the modified xylanase to carry out

catalysis, since the Vma x is decreased fourfold compared with the native enzyme. The

decrease in Km upon modification could be the result of a conformational change in the

active site caused by attachment of the pentaamminemthenkun groups.

Discussion

Initial studies have shown that oxidoreductase activity can be conferred upon

xylanases by attachment of pentaammineruthenium groups. The specific activity of the

16

modified xylanases towards the lignin monomeric substrate veratryl alcohol increases with

the number of pentaammineruthenium groups attached per molecule of xylanase.

The loss of xylanase activity observed due to modification of the B. pumilus

xylanase was unexpected, as histidine had not been reported to be a catalytically active

residue in xylanases. Two conserved glutamic acid residues had been reported to be

essential for catalysis of xylan hydrolysis in a number of xylanases. _4-_7 A comparison of

the amino acid sequences of the different xylanases indicates that three histidine residues

(positions 11, 59, and 60) are present in xynA from B. pumilus that are not conserved in

several other family G xylanases _'23 isolated from bacterial and fungal sources, including

T. reesei xyn II. The histidine at position 159 in the B. pumilus xynA is conserved among

ten bacterial and fungal xylanases, including the xylanases from B. pumilus, Bacillus

subtillus, and T. reesei. _3 Examination of the three-dimensional structure of the

xylanase 24'25reveals that His-159 is located inside the active site groove, while His-59 and

His-60 reside on a loop located below the active site. It is possible that this conserved

histidine may help carry out catalysis; thus, its modification results in a loss of hydrolytic

activity. Alternatively, attachment of the pentaammineruthenium group to a residue in the

active site cleft may obstruct access of the catalytic glutamic acid residues to the substrate.

The kinetic analysis of the reaction of native and pentaamminemthenium-modified xynA

with birchwood xylan appears to indicate that binding of the enzyme to the substrate is not

adversely affected and, in fact, would appear to be better for the modified xynA (an

approximately twofold decrease in KM). However, Vma x is decreased about fourfold for the

17

modified xynA, indicating a considerable decrease in the rate of the hydrolysis reaction.

Modification with pentaammineruthenium or other histidine-modifying reagents in

the presence or absence of xylose can be used as a probe of the role of histidines in the

active site of xylanases. A reversibly inactivated xylanase may be useful for applications

requiring strict limitation of the degree of xylan hydrolysis. A method using imidazole

instead of histidine, or a sulfhydryl reagent instead of cysteine, could be devised to increase

the cost-effectiveness of in situ reactivation of pentaammineruthenium-inactivated xylanase.

Treatment of wood pulps with xylanases to reduce the amount of chemicals required

for bleaching is a method that is currently being investigated and is of great interest to the

pulp and paper industries. 1°'26-29In one experiment, bleaching pretreatment of kraft pulps

using xylanases modified with pentaammineruthenium was shown to reduce the requirement

for chlorine dioxide to a greater degree than pretreatment with native xylanases (data not

shown). Further investigation and application of these preliminary results could, if

confirmed, result in improvement of the quality of paper while reducing the requirement

for bleaching chemicals, thus decreasing the amount ofhalogenated organic compounds that

would have to be removed from paper mill effluents. Penta_neruthenium and

pentammineruthenium-modified proteins have been reported to produce reactive oxygen

species and to carry out oxidation of organic compounds. '-3 Xylanases with attached redox

groups such as pentaammineruthenium could also be used to solubilize pulp residues and

depolymerize lignin in the treatment of effluent streams.

18

Acknowledgments

This research was sponsored by the Laboratory Directed Research and Development

Program at Oak Ridge National Laboratory, which is managed by Lockheed Martin Energy

SyStems, Inc., for the U.S. Department of Energy under contract number DE-AC05-

84OR21400. B. R. Evans is a postdoctoral research associate at Oak Ridge National

Laboratories through the Oak Ridge Institute for Science and Education at Oak Ridge

Associated Universities. The Jet Propulsion Laboratory is an operating division of the

California Institute of Technology under contract NAS7-918 with the National Aeronautics

and Space Administration.

19

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23

21. Gamerith, G., Grotcher, R., Zellinger, S., Herzog, P., and Kubicek, C. P. Cellulase-

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24

26. Ragauskas, A. J., Poll, K. M., and Cesternino, A. J. Effects of xylanase pretreatment

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Table 1. Comparison of native and 4-h pentaammineruthenium-modified xylanase xynA

Specific activity a

Xylanase

Birchwood Oat spelt Veratrylpreparation

xylan xylan alcohol

Nativeenzyme 194+ 12 137+ 11 ND

(N= 4) (N= 5)

Trial1--Mod. 19.8 14.5 0.0115

Trial2--Mod. 17.9+ 0.7 14.0+ 13.1 0.0649

(N= 2) (N= 2)

Trial 3--Mod. (+ Xylose) 148+ 1.7 86.1 + 4.2 0.00938

(N=2) (N= 3)

,,

a Specific activities for catalytic activities on the indicated substrates in pmol (mg protein) -_mill -1.

Table 2. Effect of free amino acids on xylanase activity of pentaammineruthenium(III)-

modified xynA

Amino acid added Concentration Specific activitya

(mm)

None 0 18.7+2.9

Glycine 50 17.0

Glutamicacid 50 18.1

Histidine 50 37.1

Methionine 50 20.6

Cysteine 5.0 75.1

Cysteine 10 86.0+2.1

Glutathione(Red.) 10 104.1

Glutathione(Oxid.) 10 21.7

a Specific activity in _mol reducing sugar produced (mg xylanase)-_ min-_ after incubation

for 15 min at pH 6.0 and 45°C from the substrate birchwood xylan (1%).

Figure 1 SDS gel (10-15% gradient) of xynA preparations. Lanes 1 and 6, Pharmacia

LMW markers; Lane 2, Biogel P-30 filtered xynA (Pulpzyme HBTM); Lane 3, xynA after

Biogel P-30 filtration/DEAE Sephadex ion-exchange chromatography; Lane 4, xynA

modified with 50-fold excess pentaamminerathenium for 2 h; Lane 5, xynA modified with

50-fold excess pentaammineruthenium for 4 h. Protein samples were denatured by

incubation with 2.5% SDS and 5% [3-mercaptoethanol for 5 min at 90°C.

Figure 2 Effect of pH on hydrolysis of birchwood xylan by native and

pentaammineruthenium- modified xynA. The amount of reducing sugar produced by a

reaction mixture containing 0.020 mg/ml xylanase, 1% birchwood xylan, and 50 mM

buffer was measured after 15-min incubation at 45°C. Native xynA control = o; xynA

modified for 4 h with pentaammineruthenium = O.

Figure 3 Effect of free amino acids on xylanase activity of 4-h pentaammineruthenium-

modified xynA. Native xynA control = o. Pentaammineruthenium-modified xynA was

incubated with the following amino acids: no amino acids added = · ; with 10 mM

cysteine = V; with 10 mM reduced glutathione = *; with 10 mM oxidized glutathione =

[El; with 50 mM histidine = l; with 50 mM glutamic acid = zx;with 50 mM methionine

= _; with 50 mM glycine = 9.

Figure 4 Chromatograms of the HPLC profiles of tryptic peptides from digestion of native

(_) or pentaamminemthenium-modified (--) xylanase xynA.

Figure 5 A Michaelis Menten plot of kinetic data for native and pentaammineruthenium-

modified xynA hydrolysis of birchwood xylan. Native xynA = o; 4-h

pentaammineruthenium-modified xynA = O. B Double reciprocal plot of kinetic data for

native and pentaamminemthenium-modified xynA. Native xynA = o; 4-h

pentaamminemthenium-modified xynA = O.

abbreviated running title' Pentaammine-ruthenium-modified xylanase

.... _ ....... --"'· T ................. '_r _

MW (XlO-3)

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f?ETENTtON TIME (mid

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